The invention relates to methods and means for preventing, treating or reducing inflammation by inhibiting proteolytic activity, more specifically, for preventing or reducing inflammations of skin or intestine.
Inflammations of skin (dermatitis) or intestine (enteritis) are of various origin. Initially, allergic reactions, infections with pathogenic micro-organisms, excoriation by chemical or physical means, and other causes are instrumental in causing an inflammation. These causal events are immediately followed by the necessary reaction of the body, resulting in an interplay of actions and events aiming at restoration of the skin or intestine to its original state. In this interplay of cause and effect, various activities of proteolytic enzymes are seen. Granulocyte, mast cells, macrophages and other immediate actors in inflammatory responses and attracted by cytokines to a site of inflammation, contain and secrete proteases, such as chymotryptic protease and elastase, that act as mediators or are instrumental in cleaving and removing proteins derived from pathogens or from the surrounding degenerated tissue. Bacteria, either as primary causal agent or during a secondary infection, and other pathogenic micro-organisms, secrete proteases that damage the surrounding tissue for their purposes. In this battlefield between host and invader, excess proteolytic reactions are kept at bay by, often very specific, protease inhibitors. Well known are proteinase/proteinase inhibitor systems such as PMN-elastase/alpha-1-proteinase inhibitor and cathepsin G/alpha-1-antichymotrypsin.
Proteolytic enzymes in themselves, however, can also be a cause of inflammation. This is especially the case for digestive enzymes which are found in the intestinal tract. In order to degrade dietary protein, the stomach, the pancreas and the small intestinal brush border secrete several kinds of proteases. Pepsin from the stomach works optimal at pH 2, pancreatic and brush border enzymes, such as trypsin, chymotrypsin and elastase work optimal at pH 7-8. In adults, the small intestine has a length of seven meters and the transit time of its contents is about 3 hours. This part of the intestine is colonized by only a few bacteria but is filled with a watery mixture of food and a wide array and large quantities of digestive enzymes, such as lipases and proteases. However, in the large intestine, colon and caecum, the water content is greatly reduced and the activity of the enzymes is neutralized by bacteria. Neutralized and digested remnants of food and bacteria (feces) finally leave the body via the rectum. Only when the colon cannot effectively reduce the water content and neutralize the enzymes, the feces may still contain proteolytic activity, which, during periods of diarrhoea or fecal incontinence, may be very irritating to intra-anal and perineal skin.
The skin, especially of humans, is, although it is protected by the stratum corneum which consists mainly of keratine, as any other proteinaceous substance, very susceptible to the proteolytic action of proteases. Consequently, fluid-like small intestinal content may cause severe inflammation.
In babies and infants, the intestine is much less well developed, especially the colon, and functions different from that in adults. This is the reason why digestive enzymes in feces of babies and infants are not neutralized; the contents of feces resemble more the contents of the small intestine, albeit having passed the colon. Therefore, perineal (perianal) dermatitis is more often found with babies or infants than with adults. Also, hospitalized infants and children with gastro-intestinal disorders are prone to such a dermatitis. Such a dermatitis or prunitis, defined by itchiness, skin erythema, vesiculas, wetness, edema or disruption (excoriation) of perineal skin, is also found with diaper rash and can manifest itself in rather mild to very severe forms. With diaper rash, complicating factors are the accumulation of urine, whereby ureum is converted by fecal bacteria to ammonia, thereby raising the pH to an even better value for the activity of proteolytic enzymes. Since the skin is extremely susceptible to infections, care should be taken to prevent such inflammations related to fecal proteolytic activity.
Yet other cases of dermatitis are found with patients that have undergone resections of colon and/or ileum a-stoma. Pouchitis, an intestinal inflammation, is a major complication of ileoanal anastomosis with reservoir construction after colon resection and is characterized by clinical symptoms and inflammation of the reservoir (pouch). Peristomal (circumstomal) dermatitis is found with those patients that have been provided with an ileostoma that opens up at the surface of the abdomen, ending in an artificial reservoir that needs to be emptied daily. In inflammatory bowel diseases (IBD, such as Crohn""s disease (CD), ulcerative colitis (UC) and pouchitis) and inflammation with an unknown etiology, the role of the intestinal flora and pathogens, proteolytic enzymes derived from these micro-organisms and endogenous (e.g., pancreatic or leukocyte/granulocyte) proteolytic enzymes and their contribution to degradation of protecting mucoglycoproteins and the underlying tissues, is not understood.
Especially in the above cases where the colon is removed or its function is affected or immature, it is evident that the proteolytic activity is still very high when the feces are excreted, leading to various degrees of perineal dermatitis.
It goes without saying that many medications and personal care items have been developed in order to remedy the severely itchy and often painful consequences of the above-discussed inflammations. General anti-inflammatory therapy often resorts to treatment with corticosteroids, despite the serious side-effects that are often seen with these medicaments. Other ways of treating are mainly based on providing either a protective layer to the skin, e.g., by applying a lipid-based ointment containing additives such as zinc, or by frequently cleaning an area at risk. Special personal care items have been developed, varying from specific wet wipes for perineal care, diapers that stay very dry despite heavy soiling by the child or patient, to products (stoma care appliances), such as adhesive, absorbing discs and stoma rinsing fluid that are specifically designed for stoma care patients with ileostomy or ileo-anal anastomosis.
However, none of these treatments can really do more than alleviate one or more of the above- and below-described clinical symptoms.
The invention provides a method for treating, reducing or preventing an inflammation or pruritis by subjecting a mammal to a treatment with at least one inhibitor which is capable of inhibiting proteolytic activity. Preferably, the invention provides a method whereby a protease produced or secreted, for example, granulocytes mastcells, macrophages and other actors in inflammatory processes, is inhibited. The invention is applicable to human and veterinary medicine and care.
A preferred embodiment of the invention is wherein the mammal is a human suffering from, for example, dermatitis or pruritis. Treating for example a dermatitis with a protease inhibitor reduces the proteolytic activity of the proteases involved in the inflammation pruritis. Especially when, in the interplay of causes and effects seen during inflammation, the activity of proteolytic enzymes is too high, the invention provides a method to reduce this activity (be it from host or from invader) by treatment with at least one inhibitor which is capable of inhibiting proteolytic activity.
The treatment is provided by applying the inhibitor in an ointment, cream, gel, powder, or any other suitable form to the location of the inflammation. These substances can, for example, also be carried on wipes impregnated with an inhibitor, in sprays or in rinsing fluid.
In a preferred embodiment of the invention, treatment is provided for an inflammation which is intestinal, perineal or peristomal, as is, for instance, seen with babies or infants with diaper rash, with children or adults with diarrhoea or fecal incontinence, with patients with inflammatory bowel syndrome and with stoma patients, which all suffer from the effects of proteolytic activity which is mainly fecal.
Treatment of fecal proteolytic activity can occur by applying the inhibitor in an ointment, cream, gel, powder, or any other suitable form, to the perineal or peristomal location of the inflammation. Intestinal inflammations, such as seen with IBD or pouchitis, can be treated by rinsing the affected location in the digestive tract by, for example, administering an enema, or can be administered orally, preferably in a pharmaceutical composition such as a draught or mixture pill, that can pass relatively unaffected through the esophagus and stomach.
These inhibitor substances can, for example, also be carried on wipes impregnated with an inhibitor, in sprays or in rinsing fluid. Also, it is possible to impregnate a diaper (during diaper production or shortly before use) with an inhibitor, thereby providing a method and means against diaper rash or pruritis. In a preferred embodiment, such a diaper is treated or impregnated with an inhibitor as provided by the invention in at least that diaper area (and underlying parts) that has, when in use, contact with the perineum of the baby, infant, child or adult. With diapers, the contact area normally comprises the diaper surface that is in contact with the perineum.
The invention provides a method of treatment which comprises administration to the patient or mammal prone to an inflammation of an inhibitor capable of inhibiting proteolytic activity of a protease. Inhibitors of proteolytic activity are widely known. For example, acid has an inhibiting effect on the hydrolysis of proteins by pancreatic proteases and, thus, a pH-decreasing substance can be used as an inhibitor as provided by the invention.
Also, adsorbing substances, such as activated charcoal (one such product is known as Norit), can act as a protease inhibitor through their adsorbing properties. In the experimental part, several examples are given of a treatment provided by the invention whereby activated charcoal, for example Norit(copyright), is used to treat an inflammation such as, for example, pouchitis.
In a preferred embodiment of the invention, the invention provides methods and means capable of inhibiting proteolytic activity of a protease. Many protease inhibitors are known (see, for example, G. Salvesen and H. Nagase. Proteolytic enzymes, a practical approach. Eds R. J. Beynon and J. S. Bond In: The practical approach series. 1989). Although non-specific inhibitors are known (i.e., human plasma xcex1-macroglobulin), most discriminate between protease classes or even subclasses. Substances such as peptide aldehydes or peptide chloromethyl ketones are very specific for subclasses of proteases (proteinases), depending on the peptide sequence they mimic. Others, such as metal chelators, act only against metallo-proteinases or calcium-dependent proteinases. Class-specific inhibitors are found against serine protease, cysteine protease, aspartic protease, and so on. These protease inhibitors are often commercially available as purified substances for use in biochemical preparations and may be expensive.
A preferred method according to the invention is a method wherein an inhibitor is derived from a plant, i.e., the inhibitor is a plant product comprising protease-inhibiting activity. As an example, such a product derived of a plant is activated charcoal which is obtained by burning peat or wood. A much preferred method according to the invention is a method wherein an inhibitor is derived from a plant that can give rise to fruit, seed, tubers or roots. xe2x80x9cDerivedxe2x80x9d herein, for example, comprises derived by partial purification or isolation or by obtaining the necessary genetic information and producing by modern recombinant technology known in the art.
Plants often protect their leaves, fruits, seeds, tubers or roots against pests by inclusion of potent protease inhibitors and mixtures thereof in those leaves, fruits, seeds, tubers or roots. For example, cereals and legumes, such as wheat or soy beans, contain protease inhibitors such as soy bean trypsin inhibitor (SBTI), which generally has activity against trypsine or chymotrypsin but not against other proteinase classes. Tubers and roots, such as potato and cassave, and also yam, beets, sweetroot, and others, contain potent inhibitors of a wide variety of digestive tract proteases, such as aminopeptidases, carboxypeptidases, chymotrypsin, trypsin and elastase. Because of this broad range, tuber or root-derived plant products comprising proteolytic activity according to the invention are preferred. Potato tubers are an extraordinarily rich source of a variety of inhibitors of all major intestinal digestive endo- and exo-proteinase of animals (Pearce et al., Arch. Biochem. Biophys, 213, 456-462, 1982). Such inhibitors act as anti-nutrients that are present as part of the natural chemical defense mechanisms of plants, such as tubers and roots, against attacking pests. In potatoes, major inhibitors are polypeptide trypsin inhibitor (PTI), polypeptide chymotrypsine inhibitor I and II (PCI-I and PCI-II), inhibitor II against chymotrypsin and trypsine, and carboxypeptidase inhibitor, which all have analogues in other plants. These act alone and in concert against the major animal digestive proteinases.
The invention provides the use of an inhibitor or plant product or extract capable of inhibiting proteolytic activity for preparing a pharmaceutical or personal care composition for reducing or preventing an inflammation or pruritis. In the experimental part, an example is given of such a product which comprises, potato juice or an inhibitor derived thereof, for example, by freeze-drying. Such a composition can comprise an ointment, cream, gel, powder, or any other suitable form in which an inhibitor can be applied to a patient. In a preferred embodiment of the invention, provided is the use of an inhibitor or plant product capable of inhibiting proteolytic activity for preparing a composition for reducing or preventing an inflammation or pruritis which is an intestinal, perineal or peristomal inflammation or pruritis. Such a composition can be in the form of a rinsing fluid, can be contained in capsules that pass through the esophagus and stomach, or can be in prefabricated wipes or diapers where the inhibitor (or plant product) is added during production or shortly before use. In a preferred embodiment, the invention provides the use of an inhibitor capable of inhibiting proteolytic activity for preparing a personal or medical care composition for perineal, perianal and/or peristomal care, for example to counter proteolytic activity that is faecal.
For example, it is possible to prevent perineal dermatitis by rinsing the reservoir and the perineal skin with a protease inhibitor-containing fluid or a protecting ointment with protease inhibitors. Also, it is possible to pre-treat diapers or personal care compositions that adsorb soiling with inhibitor powder.
The invention provides the use of an inhibitor or plant product capable of inhibiting proteolytic activity for preparing a pharmaceutical or personal care composition wherein the inhibitor or product is derived from a plant, preferably wherein the plant can give rise to fruit, seed, tubers or roots, such as a potato plant. Such an inhibitor (product, composition or mixture), as explained above, is active against a protease which is selected from the group of pancreatic and granulocyte proteases. In a particular embodiment of the invention, the inhibitor (composition or mixture) is capable of inhibiting papain and/or pronase, illustrating its broad spectrum and effectivity.
The invention also provides a pharmaceutical or personal care product (for example, ointments, powder, fluids) comprising inhibitors of protease activity that is capable of:
(a) preventing inflammation or pruritis-caused by feces (fecal proteases by inhibiting proteolytic enzymes from pancreatic and brush border origin), from bacterial (gutflora) origin, from leucocyte (granulocyte, mastcell, macrophage) origin in the case of inflammation of the intestine; or
(b) curing inflammation or pruritis caused by feces by, inhibiting proteases (such as elastase, cathepsins) produced by tissue (macrophages, granulocytes, mastcells); or
(c) curing skin inflammation and other diseases in which inflammation and disease activity is related to infiltrating inflammatory cells (effectorcells) and the release of proteases; or
(d) curing pruritis in general (treatment is often with antihistaminic) by local application of ointments with protease inhibitor which prevents histamine release from mastcells and protease release from fagocytes.
The invention also provides a personal care composition which can be added to rinsing fluids, wetties, powder, or ointments, for peri-anal and/or peri-stomal care or a diaper comprising an inhibitor of proteolytic activity. The invention furthermore provides a protease inhibitor for use in a method according to the invention. Attention to skin care can begin at the time of surgery, for example, inhibitor-containing rinse fluid. Inhibitors may be incorporated in stoma appliances, such as adhesive and absorbing discs and in stoma rinsing fluids and ointments.
Also, the invention provides a skin test for studying the effect of a protease inhibitor on proteolytic activity or inflammatory action of a substance, preferably of feces.
The invention is further described in the experimental part which is not meant to limit the invention.